Polyadenylate binding proteins (PABs) play important roles in gene expression. In yeast, they are involved in recruitment of large ribosomal subunits to form the translation initiation complex (1), and for the progressive shortening of polyadenylate tracts on cytoplasmic mRNAs (1). PABs also play important roles in gene expression in plants. In particular, they presumably mediate the poly(A)-cap synergism that is required for optimal translation of mRNAs in plant cells (2).
PABs are highly conserved RNA-binding proteins that specifically bind the 3' polyadenylate tracts of cytoplasmic mRNAs. PABs from different organisms have a common region in the N-terminal two-thirds of the protein that consists of four repeating RNA-binding domains of 80 to 90 amino acid residues (3). Each domain contains two conserved motifs (the so-called RNP-1 and RNP-2 motifs; 4,5). There is also a conserved domain in the C-terminal regions of PABs (6), a domain separated from the RNA binding domains by a variable spacer region. It has been suggested that the N-terminal region determines poly(A)-binding activity and the C-terminal region mediates interactions between PABs and other proteins (3,6,7).
To date, most work with plant PABs has focused on the characterizations of purified proteins and clones for PABs. Sieliwanowicz (8) described a protein in pea extracts that bound poly(A)-Sepharose and was able to stimulate translation in a cell-free system. Yang and Hunt (9) previously described the purification and characterization of a 70 kD PAB from the leaves of young pea seedlings. This protein was similar to pea PAB described by Sieliwanowicz (8) and had RNA binding properties similar to those of PABs from other organisms. Three Arabidopsis PAB genes have been isolated and characterized (6,10). One Arabidopsis gene codes for a protein with extensive similarity to PABs from other organisms but is novel in that it is expressed only in flowers (10). Another Arabidopsis PAB gene is expressed in root and shoot tissues (6), suggesting that different forms of PAB may exist in different tissues in plants. Gallie (2) inferred a positive role for plant PABs in translation, based on the increased translatability of polyadenylated RNAs in isolated tobacco protoplasts. These studies also suggested a link between the presence of a 5' cap and a 3' polyadenylate tract in an RNA, as a 5' cap was needed for the enhanced translatability provided by a 3' polyadenylate tract. This feature was not unique to plant cells, as it was also observed in Chinese hamster ovary and yeast cells (2).
As described herein, a yeast PAB gene is expressed in a plant, e.g., the yeast PAB1 gene in tobacco. The utility of this gene as a potential trans-dominant marker to probe PAB function in plants can then be assessed. A cDNA encoding a wheat PAB protein has been transformed in yeast, where it was observed to functionally complement a PAB1 mutant yeast, suggesting that this monocot protein can function in yeast (25). Apparently, however, the reverse approach, transforming a differentiated higher plant with a yeast PAB gene, has not previously been explored.